Atherosclerosis is the primary cause of cardiovascular disease, which is the most common cause of death in the United States. Atherosclerosis is characterized by the accumulation of lipids, cholesterol, calcium deposits, and cellular debris in vessel walls, and results in plaque formation, arterial obstruction, and diminished blood flow to organs. These plaques often rupture, causing myocardial infarction, stroke, or death. The main risk factors include elevated lipid levels, hypertension, and diabetes. Current treatment strategies are directed at changing patient lifestyle/diet and decreasing cholesterol via pharmacological methods. Surgical interventions with medical devices such as stents are used for advanced cases. While these therapeutic approaches have benefited many patients with this disease, they are far from ideal. One reason is that no drug or device is actually developed and tested in a model system that accurately recreates the disease being treated. Thus, there is a significant gap between early phase preclinical studies and human drug trials. The lack of an animal model that accurately replicates all of the manifestations of human atherosclerosis has been a major barrier to the development of effective therapies and interventions for this deadly disease. Several mouse models have been generated with mutations in genes important for lipoprotein metabolism, and while these models have been informative, they fail to develop the complex atherosclerotic lesions that are typical of the human disease. In contrast to mice, the physiology and anatomy of the porcine cardiovascular system closely resembles that of humans. In fact, pigs have long been used as models of cardiovascular disease, and pigs with naturally occurring mutations in their LDL receptor (LDLR) gene, and therefore possessing elevated LDL, have been reported. Although the hypercholesterolemic pig is an attractive model, the mild nature of the mutation, the high variability of the disease, the limited access by other researchers, and the expense prevent its wide use in the research community. Therefore, the ultimate goal of this project is to develop and commercialize a gene- targeted porcine model of atherosclerosis. LDLR fetal fibroblasts that we developed in Phase I will be used as nuclear donors for somatic cell nuclear transfer. Nuclear transfer embryos will be transferred to recipient females for gestation. Resulting piglets will have one targeted LDLR gene. We will characterize the LDLR- targeted pigs at the molecular and biochemical level. We will determine the lipid and lipoprotein profile in LDLR-targeted pigs and perform morphometric analysis to determine the presence and extent of atherosclerosis. Finally, we will establish breeding herds to generate LDLR-/- pigs and to expand and propagate the colony. This project will produce a porcine model of atherosclerosis that will provide academic and industry researchers with an opportunity to better understand the disease and to develop and test new therapeutics and preventative strategies. Thus, this work will accelerate the discovery of novel therapies for this costly and deadly disease. PUBLIC HEALTH RELEVANCE: This proposal specifically outlines the development, characterization and propagation of a genetically engineered porcine model of atherosclerosis. This project is relevant to the NIH's mission because it will provide a resource to stimulate discovery, therapeutic application, and the development of new diagnostic tools.